1. Field of the Invention
The present invention concerns a Multiplex Gene Regulatory ("MGR") system which involves a two component process which provides a method of gene regulation and induction in trangenic animals.
2. Background Information
Heretofore there were two basic approaches used to control transgene expression. Heretofore either inducible promoters (Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.E., Rosenfeld, M.G., Birnberg, N.C. and Evans, R.M., (1982), "Dramatic Growth of Mice that Develop from Eggs Microinjected with Metallothionein-Growth Hormone Fusion Genes", Nature, 300, 611-615; Stewart, T.A., Patttengale, P.K. and Leder, P., (1984), "Spontaneous Mammary Adenocarcinomas in Transgenic Mice that Carry and Express MTV/myc Fusion Genes, Cell, 38, 627-637; Hanahan, D., (1985), "Heritable Formation of Pancreatic B-cell Tumors in Transgenic Mice Expressing Recombinant Insulin/simian Virus 40 Oncogenes", Nature, 315, 115-122) or tissue specific promoter elements (Swift, G.H., Hammer, R.E., MacDonald, R.J. and Brinster, R.L., (1984), "Tissue-specific Expression of the Rat Pancreatic Eleastase 1 Gene in Transgenic Mice, Cell, 38, 639-646; Palmiter, R.D., Behringer, R.R., Quaife, C.J., Maxwell, F., Maxwell, I.H. and Brinster, R., (1987), "Cell Lineage Ablation in Transgenic Mice by Cell-specific Expression of a Toxin Gene", Cell, 50, 435-443); Overbeek, P.A., Chepelinsky, A.B., Khillan, J.S., Piatigorsky, J. and Westphal, H., (1985), "Lens Specific Expression and Developmental Regulation of the Bacterial Chloramphenicol Acetyltransferase Gene Driven by the Murine Alpha A-crystallin Promoter in Transgenic Mice", Proc. Natl. Acad. Sci., 82, 7815-7819) have been used to regulate genes in transgenic mice. These are single tiered regulatory systems. Regulation by either inducible or tissue specific promoters results in some level of basal transgene expression which cannot be experimentally controlled. Regulation by tissue specific promoters merely directs expression to a certain tissue or organ and does not provide a direct means for controlling gene expression.
Inducible promoters provide a method to manipulate the time of gene expressions however, this approach lacks a high degree of tissue specificity. In addition, inducible promoters are generally active during certain developmental stages. The basal activity level cannot be experimentally controlled. Thus these systems suffer from either lack of tissue specificity, endogenous basal activity (inducible promoters) or a lack of inducibility (tissue specific promoters).
Recently Jaspal S. Khillan, Keith C. Deen, Shu-hua Yu, Raymond W. Sweet, Martin Rosenberg and Heiner Wetphal, "Gene Transactivation Medicated by the TAT Gene of Human Immunodeficiency Virus in Transgenic Mice", Nucleic Acids Research, Volume 16, Number 4, 1988, 1423-1430, have used the long terminal repeat (LTR) and the tat gene of the Human Immunodeficiency Virus (HIV) to construct a two tiered regulatory system. In their system the LTR is transactivated by the tat gene product. However, the LTR-CAT transgene was active in the thymus, eye, spleen, small intestine and liver in the uninduced state. The high basal activity of the HIV LTR severely limits the utility of the Khillan et al system.
In addition to the transactivation of HIV, the tat transactivation of HTLV-1 has been introduced into mice. Transgenic mice which express the HTLV-1 tat gene is described in Michael Nerenberg, Steven H. Hinrichs, R. Kay Reynolds, George Khoury and Gilbert Jay, "The tat Gene of Human T-Lymphotropic Virus Type I Induces Mesenchymal Tumors in Transgenic Mice", Science, 237, 1324-1329, Sep. 11, 1987 and Steven H. Hinrichs, Michael Nerenberg, R. Kay Reynolds, George Khoury and Gilbert Jay, "A Transgenic Mouse Model for Human Neurofibromatosis", Science, 237, 1340-1343, Sep. 11, 1987. These two papers point out that placing viral transactivator genes in mice can yield unpredictable results, and that only some transactivator genes will be useful for controlling gene expression in transgenic non-human mammals.
Transgenic non-human mammals is the subject of U.S. Pat. No. 4,736,866 to Leder et al.
To use transgenic animals for the analysis of gene function or to produce desirable gene products it is necessary to regulate the expression of the gene in a controlled and predictable manner. Ideally one would like to control both the time and site of expression in order to maintain the highest degree of experimental and marketing flexibility. The present invention, by utilizing a two-tiered system of control, provides the necessary degree of gene regulation required to obviate the aforesaid problem.